Project Summary: While aromatase inhibitors (AI), such as letrozole, are the standard endocrine therapy of choice for postmenopausal women with early-stage metastatic estrogen-dependent breast cancer, the major limi- tation in disease management is the development of drug resistance. Unfortunately, once resistance develops, the tumors spread and become estrogen-independent, thereby limiting favorable therapeutic outcomes. The long-term goal is to better understand the mechanism(s) of AI-resistant breast cancer, in order to contribute to both the improvement of current therapeutic modalities and the development of novel treatment strategies. The objective of this application is to interrogate the mechanism(s) by which glyceollin I plus lapatinib inhibit metastasis of AI-resistant breast cancer. The central hypothesis of the proposed research is that glyceollin I and lapatinib exhibits anti-metastatic behavior in letrozole- resistant breast cancer cells by targeting genes critical for ribosomal biogenesis, proliferation and can- cer stem cell formation. The rationale for the proposed project is that identification of the mechanism(s) of action of glyceollin I and lapatinib and preliminary indications of its effectiveness in vivo will lay the groundwork for more in-depth analyses and development of effective treatment strategies for letrozole- resistant tumors. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) to delineate the mechanism(s) by which glyceollin I plus lapatinib reverse motility in letrozole-resistant breast cancer cells in vitro; 2) to elucidate the functional role of midasin in letrozole- resistant breast cancer; and 3) to interrogate the therapeutic impact of glyceollin I plus lapatinib on let- rozole-resistant metastasis in vivo. Under the first aim, letrozole-resistant cells treated with or without combination therapy will be used to measure the impact on proliferation, migration, invasion, apoptosis and cell cycle progression. Under the second aim, targeting vectors with empty, silenced or CRISPR midasin activation plasmids will be utilized to analyze nascent 60S ribosomal subunit localization, im- port/export, translation and motility in letrozole-sensitive and letrozole-resistant breast cancer cells us- ing two- and three-dimensional tissue culture approaches. Under the third aim, both an ovariectomized female nude mouse implanted with letrozole-resistant breast cancer cells treated with glyceollin I plus lapatinib and a novel human breast cancer in mouse mammary organ culture ex vivo model will be used. The approach is innovative, because (a) glyceollin I and lapatinib therapy represents a novel treatment strategy targeting growth factoring signaling, metastasis and translation (b) introduction and development of a new ex vivo model to study drug efficacy and mechanisms represents a novel and powerful tool to study tumorigenesis and metastasis after initial hormone-receptor-based therapy has failed and (c) disrupting aberrant midasin expression will regulate protein synthesis and is a crucial pathway in restoring AI sensitivity of letrozole-resistant breast cancer. This is proposed research is significant because it will elucidate the impact of midasin on AI-resistance and will be the first in the se- ries of studies potentially leading to the development of novel phytochemicals to be used as adjunctive therapy in the treatment of letrozole-refractory breast cancer.